GSM7839002: TS paused 1; Mus musculus; miRNA-Seq
Small RNA was extracted using miRNeasy kit (Qiagen, 217004).In brief, the cell pellet was homogenized in Qiazol and incubated on a benchtop for 5 min. Chloroform was added to the homogenate and was shaken vigorously for 15 sec and incubated at room temperature for 3 min. The homogenate was centrifuged at 12000 g for 15 mins at 4C.. To the aqueous layer, 70% ethanol was added and shaken vigorously. The sample was then pipetted into RNeasy Mini spin column and centrifuged at room temperature. The flow-through contains the miRNA and other small RNA. To the flow-through, 100% ethanol was added and shaken vigorously. The sample was pipetted into a RNeasy MinElute spin column (Qiagen Cat No: 74204). The column was washed with buffers RWT, RPE and 80% ethanol. Small RNAs were extracted in 14ul of RNase free water. 10 ng RNA was used as input. Small RNA libraries were prepared using the SMARTer smRNA-seq Kit for Illumina (Takara, 635031) with 12 amplification cycles followed by bead size selection (ratio 0.95x-0.7x), followed by an additional clean-up step with the bead to sample ratio of 0.95x. Library QC was done using Qubit and BioAnalyzer and the final libraries were quantified using KAPA Library Quantification Kit (Roche, 07960140001 ). Libraries were diluted to 2 nM final concentration for pooling. Pooled libraries were sequenced on a HiSeq4000 flowcell with single-end 75 bp reads to get ~15M reads per sample.